RESUMEN
The CARMA1-Bcl10-MALT1 (CBM) signalosome is a crucial module of NF-κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal-induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6-null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal-dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild-type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal-dependent regulation of CARMA1 binding to Bcl10.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor 6 Asociado a Receptor de TNF , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , FN-kappa B/metabolismo , Guanilato Ciclasa/metabolismoRESUMEN
The transcription factor NF-κB, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-κB SEs in gene control are not fully elucidated. We investigated the characteristics of NF-κB-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-κB binding sites in SE. Our study suggests that NF-κB SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions.
Asunto(s)
FN-kappa B , Factor de Transcripción ReIA , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , FN-kappa B/genética , FN-kappa B/metabolismo , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción ReIA/genética , Activación TranscripcionalRESUMEN
NF-κB is a transcription factor that activates super enhancers (SEs) and typical enhancers (TEs) and triggers threshold and graded gene expression, respectively. However, the mechanisms by which NF-κB selectively participates in these enhancers remain unclear. Here we show using mouse primary B lymphocytes that SE activity simultaneously associates with chromatin opening and enriched NF-κB binding, resulting in a higher fold change and threshold expression upon B cell receptor (BCR) activation. The higher fold change results from longer DNA, whereas the threshold response is explained by synergy in DNA-NF-κB binding and is supported by the coexistence of PU.1 and NF-κB in a SE before cell stimulation. This model indicates that the pre-existing NF-κB functions as a seed and triggers its processive binding upon BCR activation. Our mathematical modeling of the single-cell transcriptome reveals an additional role for SEs in divergent clonal responses in B cells.
Asunto(s)
Elementos de Facilitación Genéticos/genética , FN-kappa B/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Células Cultivadas , Cromatina/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Eukaryotic transcription factors (TFs) coordinate different upstream signals to regulate the expression of their target genes. To unveil this regulatory network in B-cell receptor signaling, we developed a computational pipeline to systematically analyze the extracellular signal-regulated kinase (ERK)- and IκB kinase (IKK)-dependent transcriptome responses. We combined a bilinear regression method and kinetic modeling to identify the signal-to-TF and TF-to-gene dynamics, respectively. We input a set of time-course experimental data for B cells and concentrated on transcriptional activators. The results show that the combination of TFs differentially controlled by ERK and IKK could contribute divergent expression dynamics in orchestrating the B-cell response. Our findings provide insights into the regulatory mechanisms underlying signal-dependent gene expression in eukaryotic cells.
Asunto(s)
Simulación por Computador , Regulación de la Expresión Génica , Transducción de Señal/genética , Transcripción Genética , Animales , Biocatálisis , Pollos/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Redes Reguladoras de Genes , Quinasa I-kappa B/metabolismo , Modelos Biológicos , Receptores de Antígenos de Linfocitos B/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The CARMA1/CARD11-BCL10-MALT1 (CBM) complex bridges T and B cell antigen receptor (TCR/BCR) ligation to MALT1 protease activation and canonical nuclear factor κB (NF-κB) signaling. Using unbiased mass spectrometry, we discover multiple serine phosphorylation sites in the MALT1 C terminus after T cell activation. Phospho-specific antibodies reveal that CBM-associated MALT1 is transiently hyper-phosphorylated upon TCR/CD28 co-stimulation. We identify a dual role for CK1α as a kinase that is essential for CBM signalosome assembly as well as MALT1 phosphorylation. Although MALT1 phosphorylation is largely dispensable for protease activity, it fosters canonical NF-κB signaling in Jurkat and murine CD4 T cells. Moreover, constitutive MALT1 phosphorylation promotes survival of activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) cells addicted to chronic BCR signaling. Thus, MALT1 phosphorylation triggers optimal NF-κB activation in lymphocytes and survival of lymphoma cells.
Asunto(s)
Activación de Linfocitos , Linfoma de Células B Grandes Difuso/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Antígenos CD28/metabolismo , Caseína Quinasa Ialfa/metabolismo , Células Cultivadas , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , FN-kappa B/metabolismo , FosforilaciónRESUMEN
TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex-nuclear factor-κB (NF-κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B-cell receptor (BCR) signaling, the physiological roles of TAK1 in B-cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ-positive B cells, but not for receptor editing. In contrast, NF-κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF-κB signaling in early B-cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B-cell development. TAK1-deficient mice showed significant reduction of BM Igλ-positive B-cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl-2 was reduced in TAK1-deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over-expression of exogenous Bcl-2 enhanced the survival of TAK1-deficient Igλ-positive B cells. TAK1-IKK-NF-κB signaling contributes to the survival of λ-chain-positive B cells through NF-κB-dependent anti-apoptotic Bcl-2 expression.
Asunto(s)
Linfocitos B/citología , Supervivencia Celular , Cadenas lambda de Inmunoglobulina/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Animales , Apoptosis/genética , Supervivencia Celular/genética , Células Cultivadas , Expresión Génica , Genes bcl-2 , Ratones , FN-kappa B/metabolismoRESUMEN
Transforming growth factor-ß-activated kinase (TAK)1-binding proteins (TAB) activate nuclear factor-κB by linking TAK1 to signaling molecules. We investigated the mechanisms underlying B-cell receptor (BCR) signaling in TAB2- and TAB3-deficient and TAB3 domain deletion mutant DT40 B cell lines. Loss of TAB2 and TAB3 abolished BCR-induced inhibitor of κB kinase (IKK) activation and TAK1 binding to caspase recruitment domain membrane-associated guanylate kinase protein (CARMA)1. Deletion of TAB3, coupling of ubiquitin conjugation to ER degradation, coiled-coil, and zinc finger domains blocked IKK activation and association with CARMA1. Thus, TAB2 and TAB3 connect signaling molecules that activate IKK in BCR signaling.
Asunto(s)
Quinasa I-kappa B/metabolismo , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Línea Celular , Pollos , Guanilato Ciclasa/metabolismo , Unión Proteica , Ubiquitina/metabolismoRESUMEN
Different dynamic behaviours of signalling activity can induce distinct biological responses in a variety of cells. However, the molecular mechanisms that determine the dynamics of kinase activities in immune cells are not well understood. In this study, we showed that the duration of both IκB kinase (IKK) and extracellular signal-regulated kinase (ERK) activities in B cell receptor (BCR)- and CD40-signalling pathways in B cells were regulated by transcriptional feedback loops. We conducted a time-course transcriptome analysis after BCR or CD40 stimulation and identified the following four candidate genes as feedback regulators for IKK and ERK: inhibitor of apoptosis protein (IAP), TNF alpha-induced protein 3, dual-specificity phosphatase 5, and sprouty homolog 2. Quantitative experiments and mathematical modelling suggested that IAP inhibition shortened the duration of IKK and ERK activity following both BCR and CD40 pathway stimulation, indicating a positive role for IAP in B cell signalling. Furthermore, transient kinase activities induced by IAP blockage reduced the levels of delayed expression genes. Together, our findings suggest that IKK and ERK activity durations can be fine-tuned by the coordinated regulation of positive and negative transcriptional feedback and that these network properties determine the biological output of B cells.
Asunto(s)
Linfocitos B/metabolismo , Ligando de CD40/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Línea Celular , Fosfatasas de Especificidad Dual/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Retroalimentación Fisiológica , Quinasa I-kappa B/genética , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1(-/-) mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell-independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.
Asunto(s)
Linfocitos B/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Formación de Anticuerpos , Linfocitos B/citología , Proliferación Celular , Supervivencia Celular , Endocitosis , Células HEK293 , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/metabolismo , Activación de Linfocitos , Cavidad Peritoneal/citología , Proteínas Serina-Treonina Quinasas/deficiencia , Receptores de Antígenos de Linfocitos B/metabolismo , Recombinación Genética/genética , Bazo/citología , Linfocitos T/metabolismoRESUMEN
The transcription factor nuclear factor-κB (NF-κB) signaling pathway is crucial in B-cell physiology. One key molecule regulating this pathway is the serine/threonine kinase TAK1 (MAP3K7). TAK1 is responsible for positive feedback mechanisms in B-cell receptor signaling that serve as an NF-κB activation threshold. This study aimed to better understand the correlation between TAK1-mediated signaling and B-cell development and humoral immune responses. Here we showed that a B-cell conditional deletion of TAK1 using mb1-cre resulted in a dramatic elimination of the humoral immune response, consistent with the absence of the B-1 B-cell subset. When monitoring the self-reactive B-cell system (the immunoglobulin hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model), we found that TAK1-deficient B cells exhibited an enhanced susceptibility to cell death that might explain the disappearance of the B1 subset. In contrast, these mice gained numerous marginal zone (MZ) B cells. We consequently examined the basal and B-cell receptor-induced activity of NF-κB2 that is reported to regulate MZ B-cell development, and demonstrated that the activity of NF-κB2 increased in TAK1-deficient B cells. Thus, our results present a novel in vivo function, the negative role of TAK1 in MZ B-cell development that is likely associated with NF-κB2 activation.
Asunto(s)
Linfocitos B/citología , Linfocitos B/enzimología , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Animales , Linfocitos B/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Eliminación de Gen , Inmunidad/efectos de los fármacos , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/deficiencia , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Transcription factor nuclear factor kappa B (NF-κB) shows cooperative switch-like activation followed by prolonged oscillatory nuclear translocation in response to extracellular stimuli. These dynamics are important for activation of the NF-κB transcriptional machinery, however, NF-κB activity regulated by coordinated actions of these dynamics has not been elucidated at the system level. Using a variety of B cells with artificially rewired NF-κB signaling networks, we show that oscillations and switch-like activation of NF-κB can be dissected and that, under some conditions, these two behaviors are separated upon antigen receptor activation. Comprehensive quantitative experiments and mathematical analysis showed that the functional role of switch activation in the NF-κB system is to overcome transient IKK (IκB kinase) activity to amplify nuclear translocation of NF-κB, thereby inducing the prolonged NF-κB oscillatory behavior necessary for target gene expression and B-cell activation.
RESUMEN
BACKGROUND: The CARMA1-BCL10-MALT1 (CBM) complex bridges T cell receptor (TCR) signaling to the canonical IκB kinase (IKK)/NF-κB pathway. The CBM complex constitutes a signaling cluster of more than 1 Mio Dalton. Little is known about factors that facilitate the rapid assembly and maintenance of this dynamic higher order complex. FINDINGS: Here, we report the novel interaction of the aryl hydrocarbon receptor (AHR) interacting protein (AIP) and the molecular scaffold protein CARMA1. In T cells, transient binding of CARMA1 and AIP enhanced formation of the CBM complex. Thereby, AIP promoted optimal IKK/NF-κB signaling and IL-2 production in response to TCR/CD28 co-stimulation. CONCLUSIONS: Our data demonstrate that AIP acts as a positive regulator of NF-κB signaling upon T cell activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasas/metabolismo , Guanilato Ciclasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Animales , Proteína 10 de la LLC-Linfoma de Células B , Antígenos CD28/metabolismo , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/metabolismo , Interleucina-2/metabolismo , Ganglios Linfáticos/citología , Ratones , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Complejos Multiproteicos/metabolismo , Cultivo Primario de Células , Bazo/citologíaRESUMEN
A switchlike response in nuclear factor-κB (NF-κB) activity implies the existence of a threshold in the NF-κB signaling module. We show that the CARD-containing MAGUK protein 1 (CARMA1, also called CARD11)-TAK1 (MAP3K7)-inhibitor of NF-κB (IκB) kinase-ß (IKKß) module is a switch mechanism for NF-κB activation in B cell receptor (BCR) signaling. Experimental and mathematical modeling analyses showed that IKK activity is regulated by positive feedback from IKKß to TAK1, generating a steep dose response to BCR stimulation. Mutation of the scaffolding protein CARMA1 at serine-578, an IKKß target, abrogated not only late TAK1 activity, but also the switchlike activation of NF-κB in single cells, suggesting that phosphorylation of this residue accounts for the feedback.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Guanilato Ciclasa/metabolismo , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/agonistas , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Línea Celular , Pollos , Retroalimentación Fisiológica , Guanilato Ciclasa/genética , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Noqueados , Mutación , Fosforilación , Receptores de Antígenos de Linfocitos B/genética , Serina/genética , Serina/metabolismo , Transducción de SeñalRESUMEN
The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Linfocitos T CD4-Positivos/fisiología , Guanilato Ciclasa/metabolismo , Activación de Linfocitos/fisiología , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Guanilato Ciclasa/genética , Células HEK293 , Humanos , Inmunoprecipitación , Células Jurkat , Luciferasas , Ratones , Ratones Transgénicos , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción GenéticaRESUMEN
The adaptor protein CARMA1 is required for antigen receptor-triggered activation of IKK and JNK in lymphocytes. Once activated, the events that subsequently turn off the CARMA1 signalosome are unknown. In this study, we found that antigen receptor-activated CARMA1 underwent lysine 48 (K48) polyubiquitination and proteasome-dependent degradation. The MAGUK region of CARMA1 was an essential player in this event; the SH3 and GUK domains contained the main ubiquitin acceptor sites, and deletion of a Hook domain (an important structure for maintaining inactive MAGUK proteins) between SH3 and GUK was sufficient to induce constitutive ubiquitination of CARMA1. A similar deletion promoted the ubiquitination of PSD-95 and Dlgh1, suggesting that a conserved mechanism may control the turnover of other MAGUK family protein complexes. Functionally, we demonstrated that elimination of MAGUK ubiquitination sites in CARMA1 resulted in elevated basal and inducible NF-kappaB and JNK activation as a result of defective K48 ubiquitination and increased persistence of this ubiquitination-deficient CARMA1 protein in activated lymphocytes. The coordination of degradation with the full activation of the CARMA1 molecule likely provides an intrinsic feedback control mechanism to balance lymphocyte activation upon antigenic stimulation.
Asunto(s)
Proteínas Aviares/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Linfocitos/metabolismo , FN-kappa B/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Animales , Proteínas Aviares/deficiencia , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Células Cultivadas , Pollos , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C/deficiencia , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Alineación de SecuenciaRESUMEN
Antigen receptors on the surface of B lymphocytes trigger adaptive immune responses after encountering their cognate antigens but also control a series of antigen-independent checkpoints during B cell development. These physiological processes are regulated by the expression and function of cell surface receptors, intracellular signaling molecules, and transcription factors. The function of these proteins can be altered by a dynamic array of post-translational modifications, using two interconnected mechanisms. These modifications can directly induce an altered conformational state in the protein target of the modification itself. In addition, they can create new binding sites for other protein partners, thereby contributing to where and when such multiple protein assemblies are activated within cells. As a new type of post-transcriptional regulator, microRNAs have emerged to influence the development and function of B cells by affecting the expression of target mRNAs.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Linaje de la Célula , Transducción de Señal , Inmunidad Adaptativa , Animales , Linfocitos B/metabolismo , Humanos , MicroARNs/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
The transcription factor nuclear factor-kappaB (NF-kappaB) contributes to many events in the immune system. Characterization of NF-kappaB has facilitated our understanding of immune cell differentiation, survival, proliferation, and effector functions. Intense research continues to elucidate the role of NF-kappaB, which is shared in several receptor signaling pathways, such as Toll-like receptors, the tumor necrosis factor receptor, and antigen receptors. The specificity of cellular responses emanating from stimulation of these receptors is determined by post-translational modification, or 'fine tuning', which regulates spatiotemporal dynamics of downstream signaling. Understanding the fine tuning mechanisms of NF-kappaB activation is crucial for insights into biological regulation and for understanding how cellular signaling pathways are tightly regulated to guide different cell fates. In this review, we focus on recent advances that illuminate the fine tuning mechanisms of NF-kappaB activation by BCR signaling and have increased our comprehension of complex signal systems.
Asunto(s)
Linfocitos B/metabolismo , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/citología , Humanos , Quinasa I-kappa B/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , FN-kappa B/inmunología , Proteína Quinasa C/inmunología , Proteína Quinasa C beta , Procesamiento Proteico-Postraduccional , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunologíaRESUMEN
The serine/threonine kinase MEKK3, also known as mitogen-activated protein kinase kinase kinase 3, is a critical activator of the transcription factor NF-kappaB in innate immunity. However, the physiological function of MEKK3 in adaptive immunity is unclear. Here we report that following TCR signaling, MEKK3 positively regulated the kinase, IkappaB kinase, leading to NF-kappaB activation. T cells lacking MEKK3 were defective in TCR-induced and cytokine-induced responses. Furthermore, T cell-specific deletion of MEKK3 resulted in reduced numbers of thymocytes and peripheral T cells. Thus, our results provide genetic evidence that MEKK3 plays a crucial role in adaptive immunity.
Asunto(s)
Quinasa I-kappa B/metabolismo , Activación de Linfocitos , MAP Quinasa Quinasa Quinasa 3/metabolismo , FN-kappa B/metabolismo , Linfocitos T/inmunología , Animales , Proliferación Celular/efectos de los fármacos , Quinasa I-kappa B/inmunología , Inmunidad Activa , Interleucina-2/farmacología , Interleucina-7/farmacología , MAP Quinasa Quinasa Quinasa 3/genética , MAP Quinasa Quinasa Quinasa 3/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/efectos de los fármacosRESUMEN
B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-gamma2 (PLCgamma2), Vav, B cell linker, and Bruton's tyrosine kinase in the formation of highly coordinated "microsignalosomes," the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLCgamma2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation.
Asunto(s)
Antígenos/metabolismo , Linfocitos B/citología , Linfocitos B/enzimología , Membrana Celular/metabolismo , Movimiento Celular , Fosfolipasa C gamma/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Animales , Pollos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Modelos Biológicos , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Ratas , Transducción de Señal , Quinasa Syk , Familia-src Quinasas/metabolismoRESUMEN
Protein kinase C (PKC) beta has been reported (Shinohara, H., T. Yasuda, Y. Aiba, H. Sanjo, M. Hamadate, H. Watarai, H. Sakurai, and T. Kurosaki. 2005. J. Exp. Med. 202:1423-1431; Sommer, K., B. Guo, J.L. Pomerantz, A.D. Bandaranayake, M.E. Moreno-Garcia, Y.L. Ovechkina, and D.J. Rawlings. 2005. Immunity. 23:561-574) to play a crucial role in B cell receptor (BCR)-mediated IkappaB kinase (IKK) activation through phosphorylation of caspase recruitment domain 11, Bimp3 (CARMA1). However, it remains unclear whether this PKCbeta-mediated phosphorylation accounts fully for the activation status of CARMA1, because involvement of other kinases, such as phosphoinositide 3-kinase-dependent kinase 1, has also been suggested. We show that PKCbeta mediates phosphorylation of CARMA1 on Ser668, which in turn is essential for BCR-mediated CARMA1-Bcl10-mucosal-associated lymphoid tissue 1 (MALT1) association and subsequent IKK activation. Our analyses also demonstrate that the downstream kinase IKKbeta contributes to facilitating formation of the complex CARMA1-Bcl10-MALT1 by mediating phosphorylation of CARMA1. Hence, our data suggest that PKCbeta is crucial for initial activation of IKK. The activated IKKbeta does not merely function as an effector enzyme but also modifies the upstream signaling complex through a feedback mechanism, thereby optimizing the strength and duration of the nuclear factor kappaB signal.